Dependence of stress on cross-bridge phosphorylation in vascular smooth muscle

1989 ◽  
Vol 256 (1) ◽  
pp. C96-C100 ◽  
Author(s):  
P. H. Ratz ◽  
C. M. Hai ◽  
R. A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Vascular Smooth Muscle ◽  
Light Chain ◽  
Phorbol Esters ◽  
Receptor Activation ◽  
Bay K 8644 ◽  
Cross Bridge ◽  
Cross Bridges ◽  
State Stress

Cross-bridge phosphorylation associated with agonist-stimulated contraction of vascular smooth muscle is often transiently elevated. Such observations led to the concept that phosphorylation of the 20-kDa myosin regulatory light chain (Mp) was required for initial activation and cross-bridge cycling but might not be necessary for steady-state maintenance of stress in the latch state. The possibility that stress maintenance is not regulated by phosphorylation has received some experimental support in contractions induced by phorbol esters and the calcium channel activator BAY K 8644 in which significant increases in Mp were not detected. Our aim was to test the hypothesis that phosphorylation is both necessary and sufficient for activation and for maintenance of steady-state stress. Activation of swine carotid media using agents that bypass receptor activation and elevate Ca2+ influx without mobilizing intracellular Ca2+ stores (BAY K 8644 and ionomycin) produced monotonic increases in both stress and Mp. Transient initial peaks in Mp were absent. Steady-state stress induced by both receptor- and nonreceptor-mediated activation was dependent on small increases in Mp. Increases in Mp greater than 0.3 mol Pi/mol myosin light chain had small effects on stress but produced large increases in the maximum rate of cross-bridge cycling at zero load (Vo). The experimentally determined dependence of stress on Mp was quantitatively predicted by our working hypothesis. This model proposes that Ca2+-stimulated cross-bridge phosphorylation is obligatory for cross-bridge attachment. However, dephosphorylation of attached cross bridges to form noncycling "latch bridges" allows stress maintenance with reduced Mp and cycling.

Agonist activation modulates cross-bridge states in single vascular smooth muscle cells

1993 ◽  
Vol 264 (1) ◽  
pp. C103-C108 ◽  
Author(s):  
F. V. Brozovich ◽  
M. Yamakawa
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Relative Population ◽  
Cross Bridge ◽  
Cross Bridges ◽  
State Force

To determine cross-bridge properties during agonist-stimulated contractions, steady-state force and relative steady-state stiffness were recorded at rest (pCa 9) and during both full (pCa 4) and partial (pCa 7) Ca2+ activations of isolated single alpha-toxin permeabilized vascular smooth muscle cells. For pCa 4 and pCa 7, agonist (1 microM histamine) activation resulted in significant (P < 0.05) increases in both force and stiffness. The agonist-induced increase of steady-state force was significantly (P < 0.05) greater than that of stiffness; at pCa 4, there was a 48% increase for force vs. 17% for stiffness, and, at pCa 7, there was a 160% increase for force vs. 57% for stiffness. The increase in force and stiffness after agonist prestimulation implies that the number of attached cross bridges has increased. However, after agonist prestimulation, we found that the increase of force was greater (P < 0.05) than that of stiffness, resulting in a greater force at any given level of stiffness. Thus these data indicate that agonist activation, presumably via activation of a G protein, increases the relative force per attached cross bridge, possibly by modulating the kinetics of the actomyosin adenosinetriphosphatase to increase in the relative population of cross bridges in force-producing states [actinomyosin (AM) or AM.ADP].

Regulation of shortening velocity by cross-bridge phosphorylation in smooth muscle

1988 ◽  
Vol 255 (1) ◽  
pp. C86-C94 ◽  
Author(s):  
C. M. Hai ◽  
R. A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Shortening Velocity ◽  
Internal Load ◽  
Cross Bridge ◽  
Bridge Model ◽  
The Family ◽  
Cross Bridges ◽  
Latch State ◽  
The Relationship ◽  
State Stress

We have proposed a model that incorporates a dephosphorylated "latch bridge" to explain the mechanics and energetics of smooth muscle. Cross-bridge phosphorylation is proposed as a prerequisite for cross-bridge attachment and rapid cycling. Features of the model are 1) myosin light chain kinase and phosphatase can act on both free and attached cross bridges, 2) dephosphorylation of an attached phosphorylated cross bridge produces a noncycling "latch bridge," and 3) latch bridges have a slow detachment rate. This model quantitatively predicts the latch state: stress maintenance with reduced phosphorylation, cross-bridge cycling rates, and ATP consumption. In this study, we adapted A. F. Huxley's formulation of crossbridge cycling (A. F. Huxley, Progr. Biophys. Mol. Biol. 7: 255-318, 1957) to the latch-bridge model to predict the relationship between isotonic shortening velocity and phosphorylation. The model successfully predicted the linear dependence of maximum shortening velocity at zero external load (V0) on phosphorylation, as well as the family of stress-velocity curves determined at different times during a contraction when phosphorylation values varied. The model implies that it is unnecessary to invoke an internal load or multiple regulatory mechanisms to explain regulation of V0 in smooth muscle.

Cross-bridge phosphorylation and regulation of latch state in smooth muscle

1988 ◽  
Vol 254 (1) ◽  
pp. C99-C106 ◽  
Author(s):  
C. M. Hai ◽  
R. A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Experimental Observation ◽  
Rate Constants ◽  
Regulatory Mechanism ◽  
Myosin Phosphorylation ◽  
Model Simulations ◽  
Cross Bridge ◽  
Cross Bridges ◽  
Latch State

We have developed a minimum kinetic model for cross-bridge interactions with the thin filament in smooth muscle. The model hypothesizes two types of cross-bridge interactions: 1) cycling phosphorylated cross bridges and 2) noncycling dephosphorylated cross bridges ("latch bridges"). The major assumptions are that 1) Ca2+-dependent myosin phosphorylation is the only postulated regulatory mechanism, 2) each myosin head acts independently, and 3) latch bridges are formed by dephosphorylation of an attached cross bridge. Rate constants were resolved by fitting data on the time courses of myosin phosphorylation and stress development. Comparison of the rate constants indicates that latch-bridge detachment is the rate-limiting step. Model simulations predicted a hyperbolic dependence of steady-state stress on myosin phosphorylation, which corresponded with the experimental observation of high values of stress with low levels of phosphorylation in intact tissues. Model simulations also predicted the experimental observation that an initial phosphorylation transient only accelerates stress development, with no effect on the final steady-state levels of stress. Because the only Ca2+-dependent regulatory mechanism in this model was activation of myosin light chain kinase, these results are consistent with the hypothesis that myosin phosphorylation is both necessary and sufficient for the development of the latch state.

Cyclic nucleotide dependent relaxation in vascular smooth muscle

1994 ◽  
Vol 72 (11) ◽  
pp. 1380-1385 ◽  
Author(s):  
Nancy L. McDaniel ◽  
Christopher M. Rembold ◽  
Richard A. Murphy
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Light Chain ◽  
Cyclic Nucleotide ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Regulatory Light Chain ◽  
Myosin Phosphorylation ◽  
Cross Bridge ◽  
The Relationship

Although not without controversy, the mechanisms inducing contraction of vascular smooth muscle are relatively well defined. There is a stimulus-induced increase in myoplasmic [Ca2+] with activation of myosin light chain kinase by the Ca2+–calmodulin complex, phosphorylation of the 20-kDa regulatory light chain of myosin, with subsequent cross-bridge cycling and force development. Ca2+-dependent phosphorylation of the myosin regulatory light chain appears to be the primary mechanism responsible for regulating stress in vascular smooth muscle. The relationship between myoplasmic [Ca2+] and myosin phosphorylation (i.e., the calcium sensitivity of phosphorylation) is regulated. It is higher with agonist stimulation than in tissues depolarized with high potassium solutions or after skinning procedures. The relationship between myosin phosphorylation and stress appears to be invariant with physiologic stimulation. This suggests that cross-bridge phosphorylation normally determines contraction. The mechanisms of relaxation are less well defined. In the most simple scheme, reduction of myoplasmic [Ca2+] with a fall in myosin light chain kinase activity would suffice to account for dephosphorylation of the regulatory light chain and relaxation. However, other mechanisms have been implicated in cyclic nucleotide dependent relaxation in vascular and other smooth muscle tissues. The current hypotheses of the mechanism of cyclic nucleotide dependent relaxation in vascular smooth muscle are reviewed.Key words: calcium, cyclic adenosine 3′,5′-monophosphate, cyclic guanosine 3′,5′-monophosphate, myosin light chain phosphorylation, vasodilation.

scholarly journals siRNA-mediated knockdown of h-caldesmon in vascular smooth muscle

2009 ◽  
Vol 297 (5) ◽  
pp. H1930-H1939 ◽  
Author(s):  
Elaine M. Smolock ◽  
Danielle M. Trappanese ◽  
Shaohua Chang ◽  
Tanchun Wang ◽  
Paul Titchenell ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Muscle Contraction ◽  
Light Chain ◽  
Resting State ◽  
Myosin Light Chain ◽  
Vascular Tissue ◽  
Smooth Muscle Contraction ◽  
Smooth Muscle Tissue ◽  
Cross Bridges

Smooth muscle contraction involves phosphorylation of the regulatory myosin light chain. However, this thick-filament system of regulation cannot account for all aspects of a smooth muscle contraction. An alternate site of contractile regulation may be in the thin-filament-associated proteins, in particular caldesmon. Caldesmon has been proposed to be an inhibitory protein that acts either as a brake to stop any increase in resting or basal tone, or as a modulatory protein during contraction. The goal of this study was to use short interfering RNA technology to decrease the levels of the smooth muscle-specific isoform of caldesmon in intact vascular smooth muscle tissue to determine more carefully what role(s) caldesmon has in smooth muscle regulation. Intact strips of vascular tissue depleted of caldesmon produced significant levels of shortening velocity, indicative of cross-bridge cycling, in the unstimulated tissue and exhibited lower levels of contractile force to histamine. Our results also suggest that caldesmon does not play a role in the cooperative activation of unphosphorylated cross bridges by phosphorylated cross bridges. The velocity of shortening of the constitutively active tissue and the high basal values of myosin light chain phosphorylation suggest that h-caldesmon in vivo acts as a brake against contractions due to basally phosphorylated myosin. It is also possible that phosphorylation of h-caldesmon alone in the resting state may be a mechanism to produce increases in force without stimulation and increases in calcium. Disinhibition of h-caldesmon by phosphorylation would then allow force to be developed by activated myosin in the resting state.

Halothane inhibits agonist-induced potentiation of rMLC phosphorylation in permeabilized airway smooth muscle

1997 ◽  
Vol 273 (1) ◽  
pp. L80-L85 ◽  
Author(s):  
K. A. Jones ◽  
A. Hirasaki ◽  
D. H. Bremerich ◽  
C. Jankowski ◽  
D. O. Warner
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Vascular Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Isometric Force ◽  
State Level ◽  
Steady State Level ◽  
Regulatory Myosin Light Chain ◽  
The Relationship

Agonist-induced increases in CA2+ sensitivity are mediated in part by mechanisms that increase phosphorylation of the regulatory myosin light chain (rMLC) at constant cytosolic Ca2+ concentration ([Ca2+]i). The current study tested the hypothesis that halothane inhibits acetylcholine (ACh)-induced potentiation of rMLC phosphorylation in beta-escin-permeabilized canine tracheal smooth muscle. ACh plus GTP significantly potentiated the increase in isometric force and rMLC phosphorylation induced by 0.8 microM free Ca2+. However, whereas the potentiation of isometric force was sustained, the potentiation of rMLC phosphorylation was biphasic, peaking at 0.5 min and then declining by approximately 10 min to a steady-state level significantly above that induced by 0.8 microM free Ca2+ alone. This finding suggests that mechanisms in addition to changes in rMLC phosphorylation may mediate ACh-induced Ca2+ sensitization, as has been reported for vascular smooth muscle. Halothane (0.91 +/- 0.10 mM) significantly inhibited ACh plus GTP-induced potentiation of rMLC phosphorylation and isometric force after 2 (peak rMLC phosphorylation) and 15 (steady-state rMLC phosphorylation) min of stimulation. However, the effect of halothane on the potentiation of isometric force was significantly less than that expected from its effect on rMLC phosphorylation (i.e., halothane changed the relationship between rMLC phosphorylation and isometric force). These results demonstrate that halothane inhibits the ACh-induced increase in Ca2+ sensitivity by inhibiting the membrane receptor-coupled mechanisms that increase rMLC phosphorylation at constant submaximal [Ca2+]i. Possible additional effects of halothane on rMLC phosphorylation-independent mechanisms cannot be ruled out.

Stimulus-specific changes in mechanical properties of vascular smooth muscle

1989 ◽  
Vol 257 (5) ◽  
pp. H1573-H1580 ◽  
Author(s):  
F. V. Brozovich ◽  
K. G. Morgan
Keyword(s):  
Mechanical Properties ◽  
Smooth Muscle ◽  
Steady State ◽  
Vascular Smooth Muscle ◽  
Muscle Force ◽  
State Level ◽  
Shortening Velocity ◽  
Aortic Smooth Muscle ◽  
Force Maintenance ◽  
Cross Bridges

To determine whether the mechanical properties of vascular smooth muscle are stimulus specific, force, stiffness, and the unloaded shortening velocity (Vmax) were measured during contractions of aortic smooth muscle strips stimulated with phenylephrine or KCl. After activation, muscle force and stiffness rose to a steady-state plateau where they were maintained. In phenylephrine contractions, Vmax peaked during force development and then fell to a lower steady-state level during force maintenance, whereas in the KCl contractions, Vmax did not decline during sustained contractions. Stimulation with KCl, compared with phenylephrine, produced lower steady-state forces. One possible interpretation is that the muscle formed latch cross-bridges during phenylephrine contractions, but not during KCl depolarizations. The slope of the plot of relative muscle force vs. stiffness for phenylephrine contractions, compared with KCl depolarizations, was reduced. This may imply tht the relative force per attached latch crossbridge could be reduced.

Phorbol ester-induced contraction in chemically skinned vascular smooth muscle

1986 ◽  
Vol 251 (3) ◽  
pp. C356-C361 ◽  
Author(s):  
M. Chatterjee ◽  
M. Tejada
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Phorbol Ester ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Phorbol Esters ◽  
Myosin Light Chain Phosphorylation ◽  
Kinase C

We studied the contractile response to phorbol esters and its relationship to myosin light chain phosphorylation in intact and Triton X-100-skinned porcine carotid preparations. Muscle contraction was activated by phorbol 12,13-dibutyrate (PDBu) and phorbol 12,13-didecanoate (PDD). Dose-dependent contractions to PDBu were obtained both in the intact and skinned preparations. The maximal values of stress in response to PDBu were 1.11 +/- 0.10 X 10(5) N/m2 (n = 7) in the intact and 5.72 +/- 0.59 X 10(4) N/m2 (n = 10) in the skinned muscles. The skinned tissues responded to PDD, which has been shown to activate protein kinase C, but not to the inactive isomer 4 alpha-PDD, thus ruling out nonspecific phorbol effects. The phorbol ester response exhibited a Ca2+ dependence. High stresses in the skinned muscles (5.53 +/- 0.69 X 10(4) N/m2, n = 8) were associated with low values of myosin light chain phosphorylation (0.18 +/- 0.01 mol Pi/mol light chain, n = 8). Thus phorbol esters can contract vascular smooth muscle by a mechanism that is not proportional to myosin light chain phosphorylation and that may involve activation of protein kinase C.

Protein kinase C mediation of Ca2+-independent contractions of vascular smooth muscle

1996 ◽  
Vol 74 (4) ◽  
pp. 485-502 ◽  
Author(s):  
Michael P. Walsh ◽  
Odile Clément-Chomienne ◽  
Jacquelyn E. Andrea ◽  
Bruce G. Allen ◽  
Arie Horowitz ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Light Chain ◽  
Phorbol Esters ◽  
Contractile Response ◽  
Cross Bridge ◽  
Kinase C

Tumour-promoting phorbol esters induce slow, sustained contractions of vascular smooth muscle, suggesting that protein kinase C (PKC) may play a role in the regulation of smooth muscle contractility. In some cases, e.g., ferret aortic smooth muscle, phorbol ester induced contractions occur without a change in [Ca2+]i or myosin phosphorylation. Direct evidence for the involvement of PKC came from the use of single saponin-permeabilized ferret aortic cells. A constitutively active catalytic fragment of PKC induced a slow, sustained contraction similar to that triggered by phenylephrine. Both responses were abolished by a peptide inhibitor of PKC. Contractions of similar magnitude occurred even when the [Ca2+] was reduced to close to zero, implicating a Ca2+-independent isoenzyme of PKC. Of the two Ca2+-independent PKC isoenzymes, ε and ζ, identified in ferret aorta, PKCε is more likely to mediate the contractile response because (i) PKCε, but not PKCζ, is responsive to phorbol esters; (ii) upon stimulation with phenylephrine, PKCε translocates from the sarcoplasm to the sarcolemma, whereas PKCζ translocates from a perinuclear localization to the interior of the nucleus; and (iii) when added to permeabilized single cells of the ferret aorta at pCa 9, PKCε, but not PKCζ, induced a contractile response similar to that induced by phenylephrine. A possible substrate of PKCε is the smooth muscle specific, thin filament associated protein, calponin. Calponin is phosphorylated in intact smooth muscle strips in response to carbachol, endothelin-1, phorbol esters, or okadaic acid. Phosphorylation of calponin in vitro by PKC (a mixture of α, β, and γ isoenzymes) dramatically reduces its affinity for F-actin and alleviates its inhibition of the cross-bridge cycling rate. Calponin is phosphorylated in vitro by PKCε but is a very poor substrate of PKCζ. A signal transduction pathway is proposed to explain Ca2+-independent contraction of ferret aorta whereby extracellular signals trigger diacylglycerol production without a Ca2+ transient. The consequent activation of PKCε would result in calponin phosphorylation, its release from the thin filaments, and alleviation of inhibition of cross-bridge cycling. Slow, sustained contraction then results from a slow rate of cross-bridge cycling because of the basal level of myosin light chain phosphorylation (≈0.1 mol Pi/mol light chain). We also suggest that signal transduction through PKCε is a component of contractile responses triggered by agonists that activate phosphoinositide turnover; this may explain why smooth muscles often develop more force in response, e.g., to α1-adrenergic agonists than to K+.Key words: smooth muscle, protein kinase C, calponin.

Role of myosin light-chain phosphorylation in guinea pig gallbladder smooth muscle contraction

1994 ◽  
Vol 266 (3) ◽  
pp. G469-G474 ◽  
Author(s):  
R. J. Washabau ◽  
M. B. Wang ◽  
C. Dorst ◽  
J. P. Ryan
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Smooth Muscle Contraction ◽  
Myosin Light Chain Phosphorylation ◽  
Shortening Velocity ◽  
Cross Bridges ◽  
Isotonic Shortening

In acetylcholine (ACh)-stimulated gallbladder smooth muscle, we have previously shown that phosphorylation of the 20,000-Da myosin light chains is necessary for the initiation of contraction, that myosin is stably phosphorylated at steady state, and that dephosphorylation of cross bridges is not necessary for the slowing of cross-bridge cycling rates during the period of steady-state isometric stress. The present studies were undertaken to determine whether 1) K+ (60 or 80 mM) or cholecystokinin (CCK, 10(-8) M) stimulation is accompanied by changes in myosin light-chain phosphorylation in gallbladder smooth muscle and 2) dephosphorylated noncycling cross bridges exist in K(+)- or CCK-stimulated gallbladder smooth muscle. Isometric stress, isotonic shortening velocity, and myosin light-chain phosphorylation were determined during contraction with K+ or CCK. Steady-state isometric stress was reached within 2.5 min of stimulation with K+ or CCK and was maintained for the duration of the stimulation. Stimulation with K+ or CCK was associated with rapid increases in myosin light-chain phosphorylation and maintenance of myosin light-chain phosphorylation during the stimulation. In contrast, isotonic shortening velocity was maximal at 1 min of stimulation with either K+ or CCK and then declined significantly to values that were only 26-32% of the peak velocity. These data, along with data from previous experiments with ACh, suggest that myosin light-chain phosphorylation is essential in the initiation of contraction in gallbladder smooth muscle, regardless of the source of Ca2+ or of the contractile agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

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